This invention relates to a novel bacteriocin and a putative immunity factor; they may be provided in polypeptide or nucleic acid form. Processes for using and making these products are also provided.
Bacteriocins are antibacterial peptides or proteins produced by a wide range of micro-organisms. Several of them have been isolated and characterized, and their mode of action have been determined (10–11, 16–17). They may be bacteriocidal or bacteriostatic. Of special interest are bacteriocins produced by food-grade organisms like lactic acid bacteria (e.g., Lactococcus, Streptococcus, Pediococcus, Leuconostoc, Lactobacillus, Carnobacterium) and propionic acid bacteria (e.g., Propionibacterium) because of their potential application in food preservation (29). Although a number of bacteriocins from gram-positive bacteria have broad spectrum inhibitory activity, most of the characterized bacteriocins have a relatively narrow spectrum of activity.
Antibacterial proteins produced by lactic acid bacteria have been isolated and characterized. For example, nisin is produced by strains of Lactococcus lactis and is approved as a food additive in many countries. The efficiency of nisin in preventing the growth of spoilage bacteria has been proven in a number of food systems (5). Another is pediocin PA1 produced by Pediococcus acidilactici PAC1.0 (6), which is very active against the food-borne pathogen Listeria monocytogenes (22), which is usually not very sensitive to nisin (30).
There are two major groups in the genus Propionibacterium: (i) the acnes group or cutaneous strains and (ii) the classical or dairy strains. The latter have a long history of use in dairy fermentation. These bacteria are especially important in starter cultures in the production of Swiss-type cheeses, where they are responsible for the formation of flavor, texture, and the characteristic eyes. Various MICROGARD™ MG products are produced by fermentation of skim milk with a Propionibacterium freudenreichii ssp. shermanii strain. This product inhibits molds, gram-negative species, and hetero-fermentative lactic acid bacteria. It is used as a preservative in about 30% of the cottage cheese made in the USA (3). A 700 dalton peptide has been implicated in its action, but it has not been unequivocally demonstrated that the active ingredient is a bacteriocin (7).
Among the dairy propionibacteria only two bacteriocins have been described, propionicin PLG-1 from P. thoenii P127 (13–14) and jenseniin G from P. thoenii P126 (formerly P. jensenii) (7, 24). Propionicin PLG-1 is active against a variety of micro-organisms like propionibacteria, as well as many gram-positive and gram-negative bacteria and even fungi (13). This bacteriocin has been purified to homogeneity and, according to amino acid composition analysis, it contains 99 amino acid residues and has a calculated molecular weight of 9328 (20). Propionicin PLG-1 is stable after long-term storage in dry or frozen state and kills rapidly sensitive cells upon exposure in culture medium or skim milk (9). Jenseniin G is a heat stable bacteriocin that inhibits several propionibacteria and lactic acid bacteria (24), like Lactobacillus delbruecki ssp. bulgaricus and Streptococcus thermophilus. This bacteriocin has therefore a potential role in preventing over-acidification of yogurt (31). See also U.S. Pat. Nos. 5,639,659 and 5,981,473. However, none of the bacteriocins from propionibacteria mentioned above have been thoroughly described, and their primary structures are not known.
Bacteriocin-producing starter cultures produce bacteriocins during fermentation of food products. The use of bacteriocin producers as starter cultures may therefore be of advantage in protecting fermented foods from transmission of food-borne pathogens as compared to starter cultures lacking bacteriocin. Secondary cultures containing a bacteriocin may also contribute to accelerated ripening of dairy products by killing microbes in the primary starter cultures. Dead bacteria will gradually lyse and release cell compounds like lipids, peptides, and amino acids important for the characteristics of the product. Bacteriocin-containing compositions are also applied to food surfaces in accordance with U.S. Pat. No. 6,207,210.
Several bacteriocin producers have already been isolated from foods. Therefore it is presumed that a mixture of different bacteriocins in variable amounts is already present in food products. Such bacteria and their products are generally regarded as safe (GRAS) since they have been an important part of the human diet for centuries. Microbes may be transfected with portions of the operon to produce the bacteriocin and/or the ABC transporter. One limitation to the use of bacteriocin producing propioni-bacteria is the slow growth and relatively late bacteriocin production. An alternative and more effective approach could be to use purified and concentrated bacteriocins directly as food additives.
If bacteriocins are going to be used in food manufacturing, there are some important requirements such as lack of toxicity, stability, broad activity spectrum, no effects on food properties, and a thorough understanding of their biochemical and genetic properties. Here, we describe a new bacteriocin from propionibacteria called propionicin T1. This is the first bacteriocin from propionibacteria to be characterized at the level of its amino acid and nucleotide sequences. A long-felt need to control the growth of microbes is addressed thereby. Other advantages and improvements are discussed below or would be apparent from the disclosure herein.